This invention relates to the human papillomavirus. More specifically, this invention relates to the inhibition, treatment, and diagnosis of human papillomavirus-associated lesions using synthetic oligonucleotides complementary to human papillomavirus nucleic acid.
Human papillomaviruses (HPV) comprise a group of at least 70 types, based on DNA sequence diversity as measured by liquid hybridization (Pfister et al. (1994) Intervirol. 37:143-149). These nonenveloped DNA viruses infect epithelial cells resulting in a range of lesions from benign skin and genital warts (condyloma acuminata) and epidermodysplasia verruciformis (EV) to respiratory or laryngeal papillomatosis and cervical carcinoma. Each virus type exhibits host specificity.
Several HPV types infect genital epithelia and represent the most prevalent etiologic agents of sexually transmitted viral disease. The genital HPV types can be further subdivided into xe2x80x9chigh-riskxe2x80x9d types that are associated with the development of neoplasms, most commonly HPV-16 and HPV-18; and xe2x80x9clow-riskxe2x80x9d types that are rarely associated with malignancy, most commonly HPV-6 and HPV-11. The malignant types may integrate into the genome of the host cell, thereby eliminating the requirement for viral DNA replication gene products. In contrast, the benign types, most commonly HPV6 and HPV11, rely on viral proteins E1 and E2 for replication of the episomal genome.
Current treatment for HPV infection is extremely limited. There are at present no approved HPV-specific antiviral therapeutics. Management normally involves physical destruction of the wart by surgical, cryosurgical, chemical, or laser removal of infected tissue. Topical anti-metabolites such as 5-fluorouracil and podophyllum preparations have also been used. (Reichman in Harrison""s Principles of Internal Medicine, 13th Ed. (Isselbacher et al., eds.) McGraw-Hill, Inc., NY (1993) pp.801-803). However, reoccurrence after these procedures is common, and subsequent repetitive treatments progressively destroy healthy tissue. Interferon has so far been the only treatment with an antiviral mode of action, but its limited effectiveness restricts its use (Cowsert (1994) Intervirol. 37:226-230; Bornstein et al. (1993) Obstetrics Gynecol. Sur. 4504:252-260; Browder et al. (1992) Ann. Pharmacother. 26:42-45).
Two HPV types, HPV-6 and HPV-11 are commonly associated with laryngeal papillomas, or benign epithelial tumors of the larynx. Neonates may be infected with a genital papillomavirus at the time of passage through their mother""s birth canal. By the age of two, papillomas will have developed, and infected juveniles will undergo multiple surgeries for removal of benign papillomas which may occlude the airway To date there is no method of curing juvenile onset laryngeal papillomatosis. There is consequently a serious need for a specific antiviral agent to treat human papillomavirus infectica.
New chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression (see, Zamecnik et al. (1978) Proc. Natl. Acad. Sci. (USA) 75:280-284). These agents, called antisense oligonucleotides, bind to target single-stranded nucleic acid molecules according to the Watson-Crick rule or to double stranded nucleic acids by the Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H, or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
Improved oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression. Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1988) 85:7079-7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-1 than conventional phosphodiester-linked oligodeoxynucleotides. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1989) 86:7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
In addition, chimeric oligonucleotides having ( more than one type of internucleotide linkage within the oligonucleotide have been developed. Pederson et al. (U.S. Pat. Nos. 5,149,797 and 5,220,007) discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates. Agrawal et al. (PCT US93/06884) discloses hybrid oligonucleotides having regions of deoxyribonucleotides and 2xe2x80x2-O-methyl-ribonucleotides.
A limited number of antisense oligonucleotides have been designed which inhibit the expression of HPV. For example, oligonucleotides specific for various regions of HPV E1 and E2 mRNA have been prepared (see, e.g., U.S. Pat. No. 5,364,758, WO/91/08313, WO 93/20095, and WO 95/04748).
A need still remains for the development of oligonucleotides that are capable of inhibiting the replication and expression of human papillomavirus whose uses are accompanied by a successful prognosis and low or no cellular toxicity.
It has been discovered that specific oligonucleotides complementary to particular portions of nucleic acid encoding the translational start site of human papillomavirus E1 gene can inhibit HPV replication and expression. This discovery has been exploited to provide synthetic oligonucleotides complementary to regions spanning the translational start site of mRNA encoding the HPV E1 protein.
As used herein, a xe2x80x9csynthetic oligonucleotidexe2x80x9d includes chemically synthesized polymers of about five and up to about 50, preferably from about 15 to about 30 ribonucleotide and/or deoxyribonucleotide monomers connected together or linked by at least one, and preferably more than one, 5xe2x80x2 to 3xe2x80x2 internucleotide linkage.
For purposes of the invention, the term xe2x80x9coligonucleotide sequence that is complementary to nucleic acid or mRNAxe2x80x9d is intended to mean an oligonucleotide that binds to the nucleic acid sequence under physiological conditions, e.g., by Watson-Crick base pairing (interaction between oligonucleotide and single-stranded nucleic acid) or by Hoogsteen base pairing (interaction between oligonucleotide and double-stranded nucleic acid) or by any other means, including in the case of an oligonucleotide binding to RNA, causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen base pairing under physiological conditions is measured as a practical matter by observing interference with the function of the nucleic acid sequence.
In a first aspect, the invention provides synthetic oligonucleotides complementary to a nucleic acid spanning the translational start site of human papillomavirus gene E1, and including at least 15 nucleotides. In preferred embodiments, the oligonucleotides of the invention are from about 15 to about 30 nucleotides in length.
In some embodiments, these oligonucleotides are modified. In preferred embodiments, these, modifications include at least one alkylphosphonate, phosphorothioate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, or carboxymethyl ester internucleotide linkage or a combination of such linkages, as in a chimeric oligonucleotide. In one preferred embodiment, an oligonucleotide of the invention includes phosphorothioate internucleotide linkages. In yet another preferred embodiment, the oligonucleotide includes at least one methylphosphonate internucleotide linkage.
The oligonucleotides of the invention may also include at least one deoxyribonucleotide, at least one ribonucleotide, or a combination thereof, as in a hybrid oligonucleotide. An oligonucleotide containing at least one 2xe2x80x2-O-methyl is one embodiment of the invention. In particular embodiments of the invention, the oligonucleotide has five 2xe2x80x2-O-methyls at the 3xe2x80x2 end of the oligonucleotide, or at the 3xe2x80x2 and the 5xe2x80x2 ends of the oligonucleotide. Some embodiments include at least one or at least two inosine residues at any position in the oligonucleotide.
In some embodiments, the oligonucleotides of the invention have a sequence set forth in the Sequence Listing as SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 36, 37, or 38.
In other aspects, the invention provides a pharmaceutical composition. The pharmaceutical composition is a physical mixture of at least one, and preferably two or more HPV-specific oligonucleotides with the same or different sequences, modification(s), and/or lengths. In some embodiments, this pharmaceutical formulation also includes a physiologically or pharmaceutically acceptable carrier. Specific embodiments include a therapeutic amount of a lipid carrier.
Another aspect of the invention are kits for inhibiting human papillomavirus replication and/or infection in a cell. In preferred embodiments, the kits include at least one oligonucleotide of the invention, or a combination thereof. In other preferred embodiments, at least two synthetic oligonucleotides of the invention are in the kit.
In yet another aspect of the invention, a therapeutic amount of a pharmaceutical composition containing HPV-specific synthetic oligonucleotides is administered to a cell in a method of inhibiting human papillomavirus replication. In some preferred embodiments, the method includes administering at least one oligonucleotide, or at least two oligonucleotides, having a sequence set forth in the Sequence Listing as SEQ ID NOS:1-32, 36-38, or a combination thereof.
In another aspect, a method of treating human papillomavirus infection is provided, comprising the step of administering to an infected animal or cell a therapeutic amount of a pharmaceutical composition containing at least one HPV-specific oligonucleotide, and in some embodiments, at least two HPV-specific oligonucleotides. In preferred embodiments, the oligonucleotides administered have a sequence set forth in the Sequence Listing as SEQ ID NO:1-32, 37-38, or a combination thereof.
In all methods involving the administration of oligonucleotide(s) of the invention, at least one, and preferably two or more identical or different oligonucleotides may be administered simultaneously or sequentially as a single treatment episode in the form of separate pharmaceutical compositions.
In another aspect, the invention provides a method of detecting the presence of HPV in a sample, such as a solution or biological sample. In this method, the sample is contacted with a synthetic oligonucleotide of the invention. Hybridization of the oligonucleotide to the HPV nucleic acid is then detected if the HPV is present in the sample.
Another aspect of the invention are kits for detecting HPV in a sample. Such kits include at least one synthetic oligonucleotide of the invention, and means for detecting the oligonucleotide hybridized with the nucleic acid. In a kit having more than one oligonucleotide of the invention, these oligonucleotides may have the same or different nucleotide sequences, length, and/or modification(s).